Bibliographic details of the publications referred to by the author, in this specification are collected at the end of the description.
The increasing sophistication of immunological techniques is greatly facilitating research and development in the medical, veterinary, horticultural and environmental fields. Of fundamental importance is the specificity of the antibody-antigen interaction. Through this interaction, the expression of genes encoding particular antigens can be determined.
Many disease states in avian species, plants and in particular animals, such as humans, have a genetic basis and can be characterised by changes in the patterns and/or levels of expression of various genes. For example, some cancers are associated with changes in the expression of oncogenes and tumor suppressor genes. Furthermore, disease conditions or disorders associated with changes in the cell cycle and development can be attributed to changes in transcriptional regulation of particular genes.
Although there are a number of genetic assays available to assess mutations, the identification of certain genetic changes cannot always be directly indicative of a disease condition or disorder.
Some genetic changes are expressed by alterations in cell surface antigens. Again, however, prior attempts to develop a diagnostic assay for complex disease conditions or disorders such as cancer based on the identification of a single antigen have not been universally successful.
Leukemias and lymphomas cause significant mortality and morbidity in humans. Such cancers result from the continuous proliferation of cells which would otherwise be blocked at various stages of normal differentiation to specialised cell types. Leukemias arise from blood forming cells in the bone marrow due to mutations in any of the precursors in the various lineages of differentiation (see FIG. 1). Lymphomas develop from lymphocytes or macrophages in lymphatic tissue.
Lymphocytes in the peripheral blood express a large number of different antigens on their outer plasma membranes many of which are receptors for growth factors, cell-cell interactions and immunoglobulins; molecules for cell adhesion or complement stimulation; enzymes and ion channels. A single systematic nomenclature has been developed to classify monoclonal antibodies against human leukocyte cell surface antigens known as the cluster of differentiation (CD) antigens (Kishimoto et al., 1997). Detailed information on CD antigens can be found at the website of the National Centre for Biotechnology Information (NCBI), a division of the National Library of Medicine (HLM) at the National Institutes of Health (NIH). The expression of these cell-surface antigens can distinguish different types of mature blood cells found in the peripheral circulation.
Cells in the peripheral blood are produced in the bone marrow by proliferation and differentiation down specific lineages from precursor myeloid or lymphoid stem cells which express the surface antigen CD34 (see FIG. 2).
Currently, a leukemia is diagnosed on the basis of morphology, expression of specific CD, lymphoid (LY) and myeloid (MY) antigens, enzyme activities and cytogenetic abnormalities, such as chromosome translocations. Diagnosis of acute myeloid leukemia (AML) is, for example, made using these four criteria. The expression of up to three CD antigens on leukemia cells is currently determined using fluorescently labelled antibodies against particular CD antigens with analysis by flow cytometry. Thus, screening leukemia cells from patients for expression of the 166 CD antigens is not practical using this labour intensive technique. Recently, de Matos and Vale (1996) developed a flow cytometric procedure for the simultaneous quantification in a single measurement, of the major types of human lymphocytes and their sub-sets. In this procedure, mixed populations of human lymphocytes were incubated with monoclonal antibodies against CD3, CD4, CD8, CD19 and CD56 conjugated with three different fluorochromes. Measurement of the three different fluorescent emissions by flow cytometry enables quantification of the major types of human lymphocytes, T- and B-lymphocytes and natural killer (NK) cells. Since most flow cytometers may only use three different lasers simultaneously for cell analysis, this procedure represents the maximal current use of this technique.
Chang (1983) prepared matrix-like arrays of antibodies of distinct specificities on glass cover slips which acted as minute specific immuno-adsorbants for cells expressing the corresponding antigens on their surface. This procedure was based on the prior observations that cells are able to bind to surfaces coated with antibodies specific for the antigens on the surface of cells (Mage et al, 1977; Wysocki and Sato, 1978). Chang (1983) demonstrated specific binding of peripheral blood mononuclear cells to mouse anti-human HLA-A2 antibody adsorbed to glass cover slips and mouse thymocytes to immobilized anti-Lyt 2.1 and anti-Lyt 2.2 antibodies.
In work leading up to the present invention, the inventors determined that the detection of certain binding partners can be used to diagnose disease conditions and disorders, such as cancers and non-neoplastic disorders, as well as the presence of a normal condition. The detection system can also be used to determine the presence or absence of a range of antigens and can be used to quantitate their relative amounts. This is particularly important in chemical libraries, environmental samples and in phage display libraries.